Fibrillar collagens form the largest protein structures found in complex organisms (Piez, 1984, In: Extracellular Matrix Biochemistry, Piez et al., Eds., Elsevier, New York, pp. 1–40; Prockop et al., 1995, Ann. Rev. Biochem. 64:403–434). The most abundant collagen fibrils consist almost entirely of a single monomer of type I collagen. The structure of the monomer was established several decades ago, but the precise pattern of packing of the monomer into fibrils has not been defined and remains controversial (Smith, 1968, Nature 219:157–158; Hulmes et al., 1979, Nature 282:878–880; Holmes et al., 1979, Biochem. Biophys. Res. Commun. 87:993–999; Piez et al., 1981, Biosci. Rep. 1:801–810; Hulmes et al., 1981, Proc. Natl. Acad. Sci. USA 78:3567–3571; Brodsky et al., 1982, Methods Enzymol. 82:127–173; Woodhead-Galloway, 1984, In: Connective Tissue Matrix, Hukins, Ed., Verlag Chemie, Basel, pp. 133–160; Hulmes et al., 1985, J. Mol. Biol. 184:473–477; Ward et al., 1986, J. Mol. Biol. 190:107–112; Galloway, 1985, In: Biology of Invertebrate and Lower Vertebrate Collagens, Bairoti et al., Eds., Plenum Press, New York, pp. 73–82; Chapman, 1989, Biopolymers 28:1367–1382; Jones et al., 1991, J. Mol. Biol. 218:209–219).
Type I collagen is similar to other fibrillar collagen in that it is first synthesized as a soluble procollagen containing N-propeptides and C-propeptides (Prockop et al., 1995, Ann. Rev. Biochem. 64:403–434). The propeptides are cleaved by specific N- and C-proteinases and the monomers then spontaneously assemble into characteristic fibrils. The two α1(I) chains and one α2(I) chains of a monomer of type I collagen are primarily comprised of about 338 repeating tripeptide sequences of -Gly-Xxx-Yyy- in which -Xxx- is frequently proline and -Yyy- is frequently hydroxyproline. The ends of the α1(I) and α2(I) chains consist of short telopeptides of about 11 to 25 amino acids per chain. The distribution of hydroxyproline and charged residues in the -Xxx- and -Yyy- positions in the triple-helical domain define 4.4 repeats or 4.4 D-periods of about 234 amino acids each. In longitudinal sections, the monomers are arranged in fibrils in a head-to-head-to-tail orientation with a gap of about 0.6 D-periods and, therefore, repeat of 5 D-periods. The continuity of the fibrils is maintained by many of the monomers being staggered by 1, 2, 3, or 4 D-periods relative to the nearest neighbor so as to generate gap and overlap regions. However, there are conflicting data from electron microscopy and X-ray analysis about the lateral packing of the monomers. One view is that the monomers are laterally packed in a tilted quasi-hexagonal lattice (Hulmes et al., 1979, Nature 282:878–880; Jones et al., 1991, J. Mol. Biol. 218:209–219). A related view is that the fibrils consist of “compressed” microfibrils that are comprised of monomers coiled into a rope-like pentameric structure (Smith, 1968, Nature 219:157–158; Piez et al., 1981, Biosci. Rep. 1:801–810). Still another view is that the lateral packing of the collagen in many fibrils is either liquid-like or a biological equivalent of a liquid crystal Galloway, 1985, In: Biology of Invertebrate and Lower Vertebrate Collagens, Bairoti et al., Eds., Plenum Press, New York, pp. 73–82; Chapman, 1989, Biopolymers 28:1367–1382).
One experimental approach to defining the lateral packing of the monomers was to observe the initial assembly of monomers into fibrils. Early experiments (Piez, 1984, In: Extracellular Matrix Biochemistry, Piez et al., Eds., Elsevier, New York, pp. 1–40; Ward et al., 1986, J. Mol. Biol. 190:107–112; Veis et al., 1988, In: Collagen: Biochemistry, Vol. 1, Nimni, Ed., CRC Press, Boca Raton, Fla., pp. 113–138) on the re-assembly of fibrils from collagen extracted from tissues with acidic buffers suggested that the first structures formed were linear strands of monomers bound by 0.4 D-period overlaps (4 D staggers). Other observations with extracted collagens suggested the initial stages involved assembly of structures similar to pentameric microfibrils (Piez, 1984, In: Extracellular Matrix Biochemistry, Piez et al., Eds., Elsevier, New York, pp. 1–40; Veis et al., 1988, In: Collagen: Biochemistry, Vol. 1, Nimni, Ed., CRC Press, Boca Raton, Fla., pp. 113–138; Gelman et al., 1980, J. Biol. Chem. 155:8098–8102). Subsequently, a system was developed for studying assembly of type I collagen fibrils de novo by enzymic cleavage of a purified soluble precursor of procollagen under physiological conditions (Miyahara et al., 1982, J. Biol. Chem. 257:8442–8448; Kadler et al., 1987, J. Biol. Chem. 262:15696–15701; Kadler et al., 1990, Ann. N.Y. Acad. Sci. 580:214–224; Kadler et al., 1990, Biochem. J. 268:339–343). Because thick fibrils were generated in the system, it was possible to use dark-field light microscopy to follow the growth of the fibrils through intermediate stages (Kadler et al., 1990, Biochem. J. 268:339–343). The first fibrils detected had a blunt end and a pointed tip or end. Initial growth of the fibrils were exclusively from the pointed or a-tip. Later, b-tips appeared on the blunt ends of the fibrils and the fibrils grew from both directions. Scanning transmission electron microscopy indicated that both the a-tips and b-tips were near paraboloidal in shape (Holmes et al., 1992, Proc. Natl. Acad. Sci. USA 89:9855–9859 Silver et al., 1992, Proc. Natl. Acad. Sci. 89:9860–9864). Also, it appeared that the monomers are oriented with their N-termini directed toward the tips. Subsequent experiments in the same system with type II collagen suggested that the fibrils also grew from pointed tips. However, the monomers were oriented with a C-termini directed toward the tips (Fertala et al., 1996, J. Biol. Chem. 271:14864–14869). Three different models were proposed to explain the growth of fibrils from near-paraboloidal tips. One model (Silver et al., 1992, Proc. Natl. Acad. Sci. 89:9860–9864) was based on the assumption that the initial core of the fibril was a pentameric microfibril and that the fibrinl grew by addition of monomers in a helical pattern. Simulations of the model suggested that as little as two specific binding steps were required first for assembly of the microfibrillar core and then a structural nucleus with about the same diameter as the final fibril. After assembly of the structural nucleus, the fibril grew from the paraboloidal tip by addition of monomers through only one of the two binding steps. A second and related model (Hulmes et al., 1995, Biophys. J. 68:1661–1670; Hulmes et al., 1989, J. Mol. Biol. 210:337–345) suggested that assembly began with formation of an undefined inner core and then monomers were added in spiral strands to generate the near-paraboloidal tips. The second model had the advantage that it more readily than the first model accounted for X-ray diffraction data that indicated that some fraction of monomers in fibrils were laterally packed in a tilted quasi-hexagonal lattice (Hulmes et al., 1979, Nature 282:878–880; Galloway, 1985, In: Biology of Invertebrate and Lower Vertebrate Collagens, Bairoti et al., Eds., Plenum Press, New York, pp. 73–82). In contrast to the first two models, a third model (Parkinson et al., 1994, Physical Rev. E. 50:2963–2966) was developed in which monomers were assembled by a process involving only aggregated limited diffusion. The third model, therefore, assumed that the assembly of monomers into fibrils was similar to processes such as electrochemical depositions or perhaps formation of snowflakes, and that the process did not require the presence of specific binding sites on the monomers.